A number of enzyme immunoassays (EIA) have been developed in recent years for the determination of haptens, antigens and antibodies. The basic principle of EIA is predicated on the specific binding between components of a reaction pair (e.g., antigen/antibody, hapten/antibody, etc.) wherein one component is labeled with an enzyme. The accuracy with which the enzyme activity is determined greatly effects the precision and reproducibility of EIA's.
One enzyme frequently used as a label in EIA is horseradish peroxidase. The enzyme is plentiful, inexpensive, and stable and has a high conversion rate of achromatic substrates in the presence of a peroxide to chromatic products which can be measured spectrophotometrically or fluorometrically. One disadvantage of the enzyme, however, is that substrates used to develop colored products in the presence of a peroxide can also be oxidized by air to yield the same colored species. In general, air oxidation of the substrate has an adverse effect on the reproducibility and precision of an EIA using horseradish peroxidase as a label. Although it is possible to correct, via a blank, for autooxidation of the substrate during incubation, a requirement for EIA is that the enzymatic assay be stopped before individual spectrophotometer readings are taken.
To date, investigators working in the field of enzyme immunoassays have employed sulfuric acid or sodium azide as terminating reagents for enzyme immunoassays which utilize a peroxidase label. The foregoing reagents, however, are either corrosive, toxic or pose explosions hazards. An alternative, safe method for terminating EIAs using a peroxidase label would have obvious practical advantages.